As the primary proteinaceous component of bone, skin, cartilage, basement membranes, and more, collagen serves as the molecular scaffold for animal life. Owing to the highly hierarchical nature of the extracellular matrix, the properties of collagenous supramolecular scaffolds are fundamentally defined by the complex intracellular process of collagen folding and quality control. Unsurprisingly, therefore, defects in intracellular collagen proteostasis engender diverse diseases known as the collagenopathies. These defects are most commonly caused by autosomal dominant mutations in collagen genes, and can be variously ascribed to three primary issues: (1) Escape of misfolded or dysfunctional collagen strands into the extracellular matrix; (2) Insufficient secretion of properly folded collagen; and/or (3) Intracellular accumulation of misfolding collagen molecules that leads to chronic cell dysfunction. All three of these defects are associated with a failure of the endoplasmic reticulum's (ER's) proteostasis network (a highly integrated system of chaperones, quality control mechanisms, and secretory machineries) to properly solve the collagen production problem, particularly in the context of mutations that lead to disease. Elucidating molecular mechanisms of collagen proteostasis in the ER is therefore of paramount importance to enable the development of disease- modifying therapies. To this end, the current proposal aims to answer three key questions: (1) Can collagen proteostasis defects by rescued by rational chemical biologic modulation of the ER proteostasis network? (2) How is a misfolding collagen strand identified by the ER quality control machinery? (3) How is collagen assembly, which is the critical first step in collagen folding, regulated both for wild-type collagen and for misfolding, disease-causing collagen variants? In Specific Aim 1, state-of-the-art chemical biology strategies targeted at the ER proteostasis network and the unfolded protein response are deployed to test the hypothesis that disease-associated collagen proteostasis defects can be resolved by proteostasis network modulation. In Specific Aim 2, the mechanisms of collagen quality control (which are known to exist but remain ill-defined) will be studied in detail, both for wild-type and a range of misfolding collagen variants. This Aim involves mass spectrometry-based quantitative comparative interactomics to detect such mechanisms, followed by biochemical validation and characterization. In Specific Aim 3, the molecular code for collagen assembly will be defined, and strategies to address assembly defects via the proteostasis network will be pursued. Insights obtained using this combination of biochemical and cell and chemical biological experimental strategies are expected to have a positive and ultimately translatable impact, because they are highly likely to yield new targets for therapeutic intervention in diverse collagenopathies that are not accessible by other experimental approaches.